A infectious disease in agriculture and stockbreeding/fisheries industries is one of the most important problems. Due to the enlargement of the industries such as aquaculture industry, the demand for prevention of eco-friendly disease is increasing in the current environment where the risk of infectious diseases is increasing. In particular, the discovery of drug vaccines and new materials for functional foods with increased immunity is the key technology to grow the future agriculture and stockbreeding/fisheries industries. In order to develop such vaccines for agriculture and stockbreeding/fisheries industries or functional food materials, mass production of stable and economical active substances is essential. The development of a microbial oral vaccine against animal diseases using a transgenic microorganism that produces an antigen protein available as a vaccine is not only inexpensive, but also is available for use as being added to animal feed as an oral vaccine. In this regard, it is expected that farmers will be able to increase income and reduce labor force.
Feed additives are non-nutrient supplementary substances that are assorted in small quantities for the purpose of improving productivity, and antibiotics, probiotics, enzymes, organic acids, flavors, sweeteners, antioxidants, various natural substances, and functional substances may be classified as feed additives. In reality, these feed additives are widely used in livestock feed, and when applied properly to animal feed, positive effects thereof may be resulted even in small amounts. The overall market size of the feed additives reaches about 15 trillion Korean Won in Korea, and continues to grow upon population growth, increased meat consumption, and rising grain prices. Types of the feed additives include amino acids, vitamins, growth-promoting antibiotics, minerals, enzymes, organic acids, carotenoid pigments, and preservatives. Many products are produced through a fermentation process, and their proportion is also increasing. Here, the key factor for successful feed additive business includes microbial manipulation technology and application technology of biomass.
Saccharomyces cerevisiae, which is a traditional strain, is used for fermentation of beer and bread for a long period of time, and is a generally recognized as safe (GRAS) grade microorganism whose safety to human body is guaranteed. In particular, yeast has protein secretory organelles in addition to gene transcription and translation systems that are very similar to those of higher organisms, and thus it can produce proteins that are activated by post-translational modification. In this regard, S. cerevisiae is used as a more useful host system for the mass production of medicinal proteins derived from higher organisms than Escherichia coli. In addition, since S. cerevisiae is easy to scale-up due to easy cultivation of strains and purification of extracellular secretory proteins, it is a host system useful for the industrial production of food and recombinant pharmaceutical proteins. Recombinant proteins produced using yeast strains have been successfully mass-produced with hormones (insulin, growth hormone, etc), vaccines (hepatitis vaccine, cervical cancer vaccine, etc), albumin, and hirudin.
It has been reported that beta-glucan, which is a cell wall component of yeast, acts as an adjuvant stimulating a toll-like receptor (TLR), which further increases an immune response when administered with an antigen. Afterwards, studies have been attempted to utilize S. cerevisiae, which is a traditional strain, as an antigen delivery material for oral vaccines. Recent studies have shown that, when cell wall components of yeast transmit a risk signal related to microbial infection, antigenic proteins of recombinant yeast increase activity of immune-related cells including T-cells via MHC Class I and II pathways by dendrite cells (DCs). Based on such studies, research on the possibility of developing an oral vaccine for human or veterinary use of recombinant yeast expressing an antigenic protein has attracted attention in terms of a good strategy for the development of a next-generation vaccine. In particular, from a number of studies suggest, vaccine development using recombinant yeast strain has been suggested as an effective delivery system that not only serves as an adjuvant to increase the immune response of yeast, but also appropriately targets antigens to the intestinal mucosa of host animals. In this regard, the possibility of yeast as an oral vaccine carrier is suggested.
Meanwhile, yeast is also in the spotlight as a host capable of introducing various secondary metabolite biosynthetic pathways for expression. Secondary metabolites produced by various organisms are main sources of high-value chemical compounds and often have important medicinal properties. In particular, plant metabolites prevent the infection of bacteria, viruses, and fungi through antioxidant or antibiotic functions, and are also useful as therapeutic agents because they are functional materials beneficial to human health. Thus, there is a great demand for mass production technology utilizing microorganisms. Yeast, which has its own limited secondary biosynthetic pathway, does not interfere with or compete with a foreign metabolism introduced through genetic engineering, and above all, yeast is well associated with various omics analysis systems so that it is possible to obtain comprehensive information about the physiological state of a yeast host through transcript and metabolite analysis. In addition, a detailed model regarding metabolism has been developed, and in silico yeast that can predict the behavior of the modified metabolic network has been constructed, thereby making it easier to design and manufacture artificial cells using the yeast. Furthermore, as a single-celled eukaryotic microorganism, yeast is a host suitable for the expression of a foreign enzyme, such as cytochrome P450, which becomes active only when expressed in organelles such as endoplasmic reticulum and mitochondria, and has an advantages of post-translational modification necessary for enzymatic activity derived from plants and animals, compared with a prokaryotic microbial host.
An important tool set for controlling expression of heterologous genes in yeast is a device capable of regulating the copy number of the desired expression cassette. In the case of traditional yeast, 2 micron-based plasmid includes about 5-30 copies per cell, whereas a yeast centromere/autonomously replicating sequence (CEN/ARS)-based plasmid includes a very small number of copies (about 1 copy per cell). A high-copy-number expression vector strongly expresses encoded genes, thereby generating a large burden on cells and causing instability of the expression vector itself. A low-copy-number expression vector provides a more stable expression platform, but can cause a problem with low gene expression levels. When the expression cassette is inserted into a target region on the yeast host chromosome through homologous recombination, the expression vector can be stably maintained even under the condition of no continuous selection pressure. Techniques have been developed to target ribosomal elements, delta elements, and sigma element sequences, which are present in a repetitive copy of the yeast chromosome, for multiple insertion of expression vectors.
The ribosome is composed of ribosomal RNA (rRNA) molecules and ribosomal proteins, and the eukaryotic ribosome has 28S, 16S, and 5S rRNA molecules. The eukaryotic ribosomal DNA (rDNA) is composed of a coding region and a non-coding region. In addition, rDNA is known to have an evolutionarily conserved region, an internal transcribed spacer (ITS) region showing a faster evolutionary rate than other regions, and an intergenic spacer (IGS) region. A transcription unit of the eukaryotic rRNA includes 18S, 5.8S, and 28S rRNAs in the stated order, wherein each of the rDNAs is separated by two ITS regions for the connection. Other transcription units of the eukaryotic rRNA include 5S rRNA, which is surrounded by a nontranscript sequence (NTS) site. In the case of traditional yeast, S. cerevisiae, an rDNA unit thereof is repeatedly inserted 100 to 150 times on chromosome 12. In the case of multiple insertion expression vectors using such multiple rDNA gene sites, antibiotic resistant genes are mainly used as selection markers for multiple insertion of expression cassettes. However, the use of antibiotics can cause major problems in terms of cost and environment issues as the cultivation size increases. In particular, in the case of recombinant yeast strains with antibiotic resistant markers, safety issues that can amplify the emergence of antibiotic resistant bacteria due to leakage of antibiotic resistant marker genes into ecosystem are raised as a major issue when the recombinant yeast strains are developed as oral vaccines or feed additives. Therefore, there is a need for development of an expression cassette capable of inserting a target gene in multiple copies into host chromosomes without insertion of an antibiotic resistant marker.